Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Chemistry

Program Name/Specialization

Biological and Chemical Sciences

Faculty/School

Faculty of Science

First Advisor

Dr. Anthony J. Clarke

Advisor Role

Principal investigator

Abstract

The Centres for Disease Control has acknowledged that we are entering a post-antibiotic era, where increasing numbers of bacterial pathogens are becoming resistant to known antibiotics. One of the most successfully targeted features of bacteria is also the main component of the cell wall, peptidoglycan (PG). PG is a heteropolymer of alternating sugar residues, namely N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) which are joined together by b-(1,4)-glycosidic bonds and crosslinked via stem peptide chains. PG is a target, readily lysed by exogenous and endogenous autolysins, such as lysozyme and lytic transglycosylase, respectively. This event leads to cell death if not attenuated by post-synthetic modifications, such as the O-acetylation of the C-6 hydroxyl group of MurNAc. PG O-acetylation prevents attack from lysozyme, which is an important component of the host immune response, and thus results in persistence in the host during infection. PG and its metabolic enzymes serve as effective antimicrobial targets, as PG is unique to bacteria and is required for cell viability. The enzymes responsible for PG O-acetylation in Gram-negative bacteria are PG O-acetyltransferase A and B (PatA and PatB) and they are under investigation as drug targets in Gram-negative bacterial pathogens, such as Campylobacter jejuni. While recent mechanistic insights into PG O-acetylation by PatB have been revealed, there remains no structural characterization on PatA to confirm the proposed mechanism. Many questions remain surrounding the structure of PatA and how it may interact with PatB to accomplish PG O-acetylation. In this thesis, the experimental topology of PatA from C. jejuni was determined and presented in lieu of an experimental three-dimensional structure. Through this, PatA was demonstrated to be a transmembrane protein with twelve transmembrane segments, and the positions of conserved structural and catalytic features were confirmed. Furthermore, structural modelling of PatA and a possible binding partner PatB were performed to show strong preliminary evidence for a direct protein-protein interaction between PatA and PatB, in agreement with topological and other mechanistic experiments. The data presented in this thesis provide the first structural characterization of PatA and suggest a working model for PG O-acetylation in Gram-negative pathogens as an antivirulence target.

Convocation Year

2025

Convocation Season

Spring

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